MICROPARA PREFINALS

• Recombinant DNA Technology

  Intentionally modifying genomes of organisms, by natural and artificial processes, for practical purposes
• Three goals of Recombinant DNA Technology

  • Eliminate undesirable phenotypic traits in humans, animals, plants, and microbes
  • Combine beneficial traits of two or more organisms to create valuable new organisms
  • Create organisms that synthesize products that humans need
• Mutagens

  Physical and chemical agents that produce mutations
• How scientists utilize mutagens

  1. Create changes in microbes' genomes so phenotypes are changed

  2. Select for and culture cells with beneficial characteristics
• Today, mutated genes alone can be isolated
• Restriction Enzymes

  Bacterial enzymes that cut DNA molecules only at specific locations (restriction sites)
• Types of restriction enzyme cuts

  • Cuts with sticky ends
  • Cuts with blunt ends
• Vectors

  Nucleic acid molecules that deliver a gene into a cell
• Useful properties of vectors

  • Small enough to manipulate in a lab
  • Survive inside cells
  • Contain recognizable genetic marker
  • Ensure genetic expression of gene
• Examples of vectors

  • Viral genomes
  • Transposons
  • Plasmids
• Gene Libraries

  A collection of bacterial or phage clones; each contains a portion of the genetic material of interest
• Typically, each clone in library contains one gene of an organism's genome
• Library may contain all genes of a single chromosome
• Library may contain set of cDNA complementary to mRNA
• Polymerase Chain Reaction (PCR)

  1. Denaturation

  2. Priming

  3. Extension
• PCR can be automated using a thermocycler
• Tools and Techniques of Recombinant DNA Technology

  • Mutagen
  • Reverse transcriptase
  • Synthetic nucleic acid
  • Restriction enzyme
  • Vector
  • Gene Library
  • Polymerase chain reaction (PCR)
  • Gel electrophoresis
  • Electroporation
  • Protoplast fusion
  • Gene gun
  • Microinjection
  • Southern blot
  • Nucleic acid probes
  • Genetic mapping
  • DNA sequencing
  • DNA microarray
• Potential Applications of the Tools and Techniques

  • Creating novel genotypes and phenotypes
  • Synthesizing a gene using an mRNA template
  • Creating DNA probes to localize genes within a genome
  • Creating recombinant DNA by joining fragments
  • Altering the genome of a cell
  • Providing a ready source of genetic material
  • Multiplying DNA for various applications
  • Separating DNA fragments by size
  • Inserting a novel gene into a cell
  • Identifying a strain of pathogen
  • Localizing specific genes in a Southern blot
  • Comparing genomes of organisms
  • Diagnosis of infection
• Must find clone containing DNA of interest when selecting a clone of recombinant cells
• Probes are used to find the clone containing the DNA of interest
• Genetic Mapping


  Locating genes on a nucleic acid molecule
• Genetic mapping provides useful facts concerning metabolism, growth characteristics, and relatedness to others
• Most microorganisms have never been grown in a laboratory, so scientists know them only by their DNA fingerprints
• DNA fingerprints have allowed identification of over 500 species of bacteria from human mouths
• DNA fingerprints have determined that methane-producing archaea are a problem in the production of methane by rice agriculture
• Pharmaceutical and Therapeutic Applications of Recombinant DNA Technology

  • Protein Synthesis
  • Vaccines
  • Genetic screening
  • DNA fingerprinting
  • Gene therapy
  • Medical diagnosis
  • Xenotransplants
• Some Products of Recombinant DNA Technology Used in Medicine

  • Interferons
  • Interleukins
  • Tumor necrosis factor
  • Erythropoietin
  • Tissue plasminogen activating factor
  • Human Insulin
  • Taxol
  • Factor VIII
  • Macrophage colony stimulating factor
  • Relaxin
  • Human Growth Hormone
  • Hepatitis B vaccine
• Agricultural Applications of Recombinant DNA Technology

  • Production of transgenic organisms
  • Herbicide resistance
  • Salt tolerance
  • Freeze resistance
  • Pest resistance
  • Improvements in nutritional value and yield
• Plasmids


  Small molecules of DNA that replicate independently, carry information required for their own replication, and often for one or more cellular traits, and are not essential for normal metabolism, growth, or reproduction but can confer survival advantages
• Types of plasmids


  • Fertility factors
  • Resistance factors
  • Bacteriocin factors
  • Virulence plasmids
  • Cryptic plasmids
• Some fungi and protozoa carry plasmids
• Characteristics of Microbial Genomes

  • Number of Chromosomes
  • Plasmids present?
  • Type of nucleic acid
  • Location of DNA
  • Histones present?
• DNA Replication

  1. Requires monomers and energy

  2. Complementary structure of the two strands is key

  3. Semiconservative - new strands composed of one original strand and one daughter strand
• Genotype

  Set of genes in the genome
• Phenotype

  Physical features and functional traits of the organism
• The Transfer of Genetic Information

  1. Transcription - information in DNA is copied as RNA nucleotide sequences

  2. Translation - polypeptides synthesized from RNA nucleotide sequences
• Central dogma of genetics: DNA transcribed to RNA, RNA translated to form polypeptides
• Some Exceptions to the Genetic Code

  • AUA codes for methionine in mitochondria instead of isoleucine
  • UAG codes for glutamine in some protozoa and algae and for pyrrolysine in some prokaryotes instead of STOP
  • CGG codes for tryptophan in plant mitochondria instead of arginine
  • UGA codes for tryptophan in mitochondria and mycoplasmas instead of STOP or selenocysteine
• Comparison of Genetic Processes

  • Replication - to duplicate cell's genome, beginning at origin, ending at origin or end of linear DNA
  • Transcription - to synthesize RNA, beginning at promoter, ending at terminator
  • Translation - to synthesize polypeptides, beginning at AUG start codon, ending at UAA, UAG, or UGA stop codons
• Regulation of Genetic Expression

  75% of genes are expressed at all times, 25% in cancer cells, other genes regulated to be transcribed and translated only when needed to conserve energy, typically by halting transcription or stopping translation directly